Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells
Introduction
Optical imaging is a flexible method for studying various cellular activities. Organic dyes have been developed that can faithfully report many biological variables including calcium concentration, pH, or membrane potential (Brown et al., 1975, MacDonald and Jobsis, 1976, Davila et al., 1973). These optical probes enable simultaneous measurements from many locations and have been used to study the physiology of single neurons as well as large populations of cells. For example, calcium dye staining of the olfactory sensory neurons has enabled the mapping of the input signal to the olfactory bulb in response to odors (Wachowiak and Cohen, 2001).
Two major drawbacks of these organic dyes are the indiscriminate cell staining and the low accessibility of the dye to some cell types. To better understand the information processing that occurs in the olfactory bulb, for example, one would need to compare the neuronal output conveyed by the mitral cells to the input maps of the olfactory sensory neurons. We have begun to investigate genetically encoded voltage sensitive probes (FP-voltage sensors) because in a transgenic animal a genetically encoded sensor could in principle be expressed in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression.
The first FP-voltage sensor, denoted FlaSh, was obtained by inserting GFP downstream of the pore region in the Drosophila voltage-gated potassium channel, Shaker (Siegel and Isacoff, 1997). When FlaSh was expressed in Xenopus oocytes, changes in membrane potential were reported by changes of its fluorescence. The rate of rise of the fluorescence response varied from approximately 10 to hundreds of milliseconds, depending on which variant of GFP was used; they were relatively slow compared to an action potential (Guerrero et al., 2002). Ataka and Pieribone (2002) inserted GFP into the rat skeletal muscle voltage-gated sodium channel and obtained a faster probe. This construct (SPARC) has a smaller fluorescence signal but follows changes in membrane potential with a time constant of less than 1 ms when expressed in oocytes. A third voltage sensitive fluorescent probe, VSFP-1, has been shown to give a signal when stably expressed in mammalian HEK 293 cells (Sakai et al., 2001). VSFP-1 utilizes fluorescence resonance energy transfer (FRET) with the donor CFP and the receptor YFP fused in tandem downstream of the S4 helix of the potassium channel, Kv1.2.
To examine their suitability for use in a transgenic mouse we tested Flare (a Kv1.4-YFP variant of FlaSh), VSFP-1, and SPARC by transient expression in HEK 293 cells and dissociated hippocampal neurons. Using whole cell voltage clamp, we were unable to detect an optical signal (after averaging 16 trials) with any of the constructs in response to changes in membrane potential in either cell type. The organic voltage sensitive dye, di8-ANEPPS, was used as a control in the optical recording measurements. di8-ANEPPS also served as a cell surface marker to determine the plasma membrane expression of the FP-voltage sensors (Zimmer et al., 2002a, Zimmer et al., 2002b). All three FP-voltage sensors exhibited predominantly intracellular staining in the mammalian cells. In contrast, the voltage-gated potassium channel Kv1.4 with an N-terminal GFP (Kv1.4-N-GFP) and the sodium/potassium/chloride cotransporter with YFP inserted near the carboxy-terminus, NKCC1-YFP, exhibited mainly plasma membrane expression.
Section snippets
Expression of FP-voltage sensors in mammalian cells
E18 hippocampi (Brain Bits, Springfield, IL) were dissociated as described by Brewer and Price (1996). Dissociated neurons and HEK 293 cells were plated onto poly-l-lysine coated coverslips. Transient transfections using lippofectamine 2000 (Invitrogen) were carried out following the manufacturer's instructions. The HEK 293 cell line stably expressing NKCC1-YFP was a gift from Biff Forbush and Meike Pederson. The plasmid encoding an N-terminal GFP Kv1.4 protein (Kv1.4-N-GFP) was given to us by
Optical signals in HEK 293 cells
To measure voltage dependent fluorescence signals, whole cell voltage clamp was performed on HEK 293 cells expressing an FP-voltage sensor. As a control, we carried out similar measurements on cells stained with the organic voltage sensitive dye, di8-ANEPPS, or cells expressing an N-terminal fusion of GFP and the Kv1.4 potassium channel (Kv1.4-N-GFP). The membrane potential was held at −70 mV and subjected to a 20 ms −50 mV hyperpolarizing pulse followed by +50 mV and +100 mV depolarizing pulses (
Discussion
Three FP-voltage sensors (Flare, SPARC, and VSFP-1) were expressed in mammalian cells to determine their suitability for transgenic expression in mammals. No optical signals were detected in the average of 16 trials for any of the FP-voltage sensors when transiently expressed in HEK 293 cells or in dissociated hippocampal neurons. A fractional fluorescence change, ΔF/F, of ∼0.5% could have been detected. In the same preparations signals from the organic voltage sensitive dye, di8-ANEPPS, were
Acknowledgements
We thank Reiko Fitzsimmons, Teresa Giraldez, Maja Djurisic, Dejan Zecevic and Victor Pantani for assistance with patch clamping. We also thank Megan Baker and Christina D’Agata for their help with cell culture and constructive criticisms. Supported by NIH grants DC05259, NS050833, and R21MH064214 and HFSP grant RGP23.
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Aristotle University, School of Biology, Dept. of Zoology, Lab. Animal Physiology, Thessaloniki 54124, Greece.